Agent promoting the formation of skin basement membrane, agents promoting the formation of artificial skin and process for producing artificial skin

ABSTRACT

The present invention discloses an artificial skin formation promoting agent and skin basement membrane stabilizing agent comprising matrix metalloproteinase inhibitor or matrix metalloproteinase inhibitor and matrix protein production promoting agent; as well as a production method of artificial skin comprising adding matrix metalloproteinase inhibitor or matrix metalloproteinase inhibitor and matrix protein production promoting agent to an artificial skin formation medium.

FIELD OF THE INVENTION

[0001] The present invention relates to a skin basement membranestabilizing agent. In addition, the present invention relates toartificial skin formation promoting agent and a production method forartificial skin.

BACKGROUND ART

[0002] In the field of cosmetics and dermatology, a wide variety ofmeasures have been proposed and attempted to alleviate or treat injuriescaused by the effects of the external environment and aging includingthe exposure of the skin to sunlight. For example, examples of skinchanges accompanying aging primarily include the formation of wrinkles,and hardening or decreased resiliency.

[0003] Primary interest is being directed at decreased function ofcollagen fibers and elastic fibers composed of collagen, elastin andglucosaminoglycans in the-skin dermis as the cause of such changes. Inthe past, the use of hydroxycarboxylic acids (e.g., Japanese Patent No.2533339), and the use of lysophospholipids (e.g., Japanese UnexaminedPatent Publication No. 8-67621) or that described in the Journal of theJapan Society of Fats and Oils, Vol. 46, No. 9 (1997), pp. 13-19 havebeen proposed as means for preventing or repairing such changes.

[0004] In the former publication, keratin and wrinkles have beensuggested as being able to be eradicated by preventing decreases incollagen fibers. On the other hand, in the latter publication,lysophospholipids are suggested to demonstrate whitening effects byaccelerating the production ability of glycosaminoglycans (and morespecifically, hyaluronic acid) in human fibroblasts.

[0005] The most powerful effect of the above external environment onskin aging is produced by ultraviolet rays present in sunlight, andthese ultraviolet rays have clearly been established to be a factor thatpromotes aging. Ultraviolet rays are known to induce skin changesreferred to as photoaging that is characterized by deep wrinkles(Scharffetter-Kochanek, Advances in Pharmacology, 1997, 58, 639-655).Ultraviolet rays have a diverse range of effects on the skin, includingdamage to genetic DNA, induced production of active oxygen, and morerecently, induced production of matrix-metalloproteinases (Fisher, etal., Nature, 1996, 379, 335-339).

[0006] Due to the multifunctional nature of ultraviolet rays, themechanism by which this photoaging induced by ultraviolet rays occurshas not been adequately elucidated. Deep wrinkles corresponding to humanphotoaged skin have been clearly shown to form on the skin on the backsof hairless mice following continuous irradiation of ultraviolet rays atan energy level not enough to cause erythema. Substances having aneffect on wrinkles have also been evaluated using this mouse model(Moloney, et al., Photochem. Photobiol. 1992, 56, 495-504). However, thewrinkle formation mechanism has still not been adequately elucidated andits elucidation is awaited.

[0007] On the other hand, Koivukangas, et al. reported in 1994 thatlevels of gelatinase, an enzyme that decomposes the basement membrane,becomes elevated in skin irradiated with ultraviolet rays (Acta Derm.Venereol. 1994, 74, 279-282). In addition, it has also been reportedthat the basement membrane exhibits structural changes at sites of theskin exposed to sunlight, and that reduplication is observedparticularly frequently (Lavker, J. Invest. Dermal. 1979, 73, 59-66).This suggests the possibility that ultraviolet rays contained insunlight have an effect on basement membrane structure by increasing theamount of basement membrane decomposing enzymes produced in the skin.

[0008] However, there is no specific means known for promoting theformation of skin basement membrane structure.

[0009] Artificial skin is important as an alternative to natural skinthat has been damaged by some cause, or as an experimental material fortesting the action and pharmacological effects of pharmaceuticals andcosmetics on the skin. In either of these applications, artificial skinis desired that has a structure that imitates the structure of naturalskin as closely as possible.

[0010] Natural skin is roughly composed of the epidermis, dermis andbasement membrane present between them. One example of a knownproduction method of artificial skin that imitates natural skin involvesthe formation of an epidermal layer by culturing normal human epidermalkeratinocytes on contracted type I collagen gel having a structure thatresembles the dermis and contains human fibroblasts. In this method,however, there was the problem of inadequate formation of a basementmembrane between the collagen gel imitating the dermis and the epidermallayer imitating the epidermis.

DISCLOSURE OF THE INVENTION

[0011] Thus, the object of the present invention is to provide a novelmeans for stabilizing skin basement membrane, and to provide a novelmeans for achieving adequate formation of basement membrane in theproduction of artificial skin.

[0012] As a result of various studies to solve the above problems, theinventors of the present invention found that the formation of a skinbasement membrane structure is promoted by administering matrixmetalloproteinase inhibitor, or both matrix metalloproteinase inhibitorand matrix protein production promoting agent, thereby leading tocompletion of the present invention.

[0013] Thus, the present invention provides a skin basement membraneformation promoting agent that contains matrix metalloproteinaseinhibitor.

[0014] In addition, the present invention provides a skin basementmembrane formation promoting agent containing matrix metalloproteinaseinhibitor and matrix protein production promoting agent.

[0015] As a result of various studies to solve the above problems, theinventors of the present invention found that, in culturing to produceartificial skin, formation of a basement membrane can be promoted byadding matrix metalloproteinase inhibitor, or both matrixmetalloproteinase inhibitor and matrix protein production promotingagent to the medium, thereby leading to completion of the presentinvention.

[0016] Thus, the present invention provides an artificial skin formationpromoting agent containing matrix metalloproteinase inhibitor.

[0017] In addition, the present invention provides an artificial skinformation promoting agent containing matrix metalloproteinase inhibitorand matrix protein production promoting agent.

[0018] In addition, the present invention provides an artificial skinproduction method comprising the addition of matrix metalloproteinaseinhibitor to an artificial skin formation medium.

[0019] Moreover, the present invention provides an artificial skinproduction method comprising the addition of matrix metalloproteinaseinhibitor and matrix protein production promoting agent to an artificialskin formation medium.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a photograph representing a biological form of thecross-section of artificial skin comparing the case of addition ofmatrix metalloproteinase CGS27023A (10 μM) or MMP inhibitor (300 μM)with a control to which neither is added in culturing for the formationof artificial skin.

[0021]FIG. 2 is a photograph representing a biological form of thecross-section of artificial skin that shows the results of performinghematoxylin and eosin staining (H & E) and immunostaining (detection oflaminin (LN5)) on formed artificial skin in the case of adding matrixmetalloproteinase inhibitor CGS27023A (shown as CGS) only, the case ofadding CGS and transforming growth factor α, a matrix protein productionpromoting agent (CGS+TGFa), the case of adding CGS and transforminggrowth factor β1, a matrix protein production promoting agent(CGS+TGFb1), and the case of adding both TGFa and TGFb1 to CGS(CGS+TGFa+TGFb1) to the medium during culturing for formation ofartificial skin.

[0022]FIG. 3 is a photograph used in place of a drawing representing abiological form of a fragment of artificial skin showing the results ofperforming immunostaining (detection of type IV collagen (IVC) anddetection of type VII collagen (VIIC)) in the same experiment as FIG. 2.

[0023]FIG. 4 is a photograph representing a biological form ofcross-sections of artificial skin formed in the case of adding matrixmetalloproteinase inhibitor CGS27023A and soy bean lecithin, a matrixprotein production promoting agent (CGS+soy bean lecithin), and the caseof not adding the above substances (control) to the medium duringculturing for the formation of artificial skin.

EMBODIMENTS FOR CARRYING OUT THE INVENTION Matrix MetalloproteinaseInhibitor

[0024] There are no particular restrictions on the matrixmetalloproteinase inhibitor used in the present invention, and anysubstance may be used provided it has such inhibitory activity. Examplesof matrix metalloproteinase include gelatinase, collagenase, stromelysinand matrilysin. Thus, a substance (inhibiting) such as gelatinase,collagenase, stromelysin or matrilysin can be selected for the matrixmetalloproteinase inhibitor.

[0025] Specific examples of matrix metalloproteinase inhibitors includesubstance CGS27023A (N-hydroxy-2-[[(4-methoxyphenyl)sulfonyl]3-picolyl)amino]-3-methylbutane amide hydrochloride) (J. Med.Chem. 1997, Vol. 40, p. 2525-2532), and MMP-inhibitor(p-NH₂-Bz-Gly-Pro-D-Leu-Ala-NHOH) (FN-437) (BBRC, 1994, Vol. 199, p.1442-1446).

[0026] Moreover, specific examples of metalloproteinase inhibitor of thepresent invention that can be used include various plant extracts andpurified products obtained therefrom. Examples of such plants includeThymus serpyllum L., Valeriana faureie Briquet or other similar plants(Valerianaceae), Diospyros kaki Thunberg (Ebenaceae), Astragalus sinicusLinne (Leguminosae), Crataegus cuneata Siebold et Zuccarini (Rosaceae),Paeonia suffruticosa Andrews (Poeonia montan Sims) (Paconiaceae), Theasinensis Linne var. assamica Pierre (Thcaccae), Eucalyptus globulusLabillardiere or its similar plants (Myrtaceae), Potentilla tormentillaSchrk (Rosaceae), Tilia corda:a Mill., Tilia platyphyllus Scop., Tiliaeuropaea Linne (Tiliaceae), Betula alba Linne (Betulaceze),Origanummajorana L., Uncaria gambir Roxburgh (Rubiaceae), Juglans regiaLinne var. sinensis De Candolie or its similar plants (Juglandaceae),Sophora flavescens Aiton (Leguminosae), Sanguisorba officinalis Linne(Rosaceae), Hypericum perforatum Linne or Hypericum erectum Thunberg(Guttiferae), Thea sinensis Linne (Theaceae), Curcuma longa L(Zingiberaceae), purified extracts of Curcuma longa L includingSymplocos racemosa and Cyperus rotundus, Cyperus scariosus, Gaultheriafragrantissima, Acacia fornensia, Terminalia chebula, Ficus bengalensis,Cassia fistula Linn, Lyonia ovalifolia, Calophylium inophyllum and Ficusreligiosa.

[0027] Extracts of these plants are obtained from the root, leaves, stemor flower in the case of herbaceous plants, or from the root, buds,bark, fruits, leaves or flowers in the case of woody plants.

[0028] Extracts from these plants are obtained by drying the plantmaterial as necessary and cutting or crushing as necessary, followed byextracting into an aqueous extracting agent or organic solvent. Examplesof aqueous extracting agents that can be used include cold water, warmwater and hot water at the boiling point or lower temperature, whileexamples of organic solvents that can be used include methanol, ethanol,1,3-butanediol and ether at normal temperature or heated.

[0029] Examples of matrix proteins in the present invention includelaminin, type IV collagen, type VII collagen, parlecan and nidogen,which are constituent components of the basement membrane.

Matrix Protein Production Promoting Agent

[0030] Examples of the matrix protein production promoting agent used inthe present invention include, in the case of promoting the productionof these proteins, soy bean lysolecithin, transforming growth factor α(TGFa), transforming growth factor β1 (TGFb1), transforming growthfactor β2 (TGFb2), transforming growth factor β3 (TGFb3) and epitheliumgrowth factor (EGF).

[0031] In the case the artificial skin formation promoting agent or skinbasement membrane stabilizing agent of the present invention containsmatrix metalloproteinase inhibitor and matrix protein productionpromoting agent, the ratio of matrix metalloproteinase inhibitor andmatrix protein production promoting agent, although varying according totheir types and activity, is roughly 1:10⁶ to 10⁶:1.

Artificial Skin

[0032] Any arbitrary medium that is conventionally used for theproduction of artificial skin can be used for the basal medium used toproduce artificial skin in the present invention, and examples of suchmedia include Dulbecco's Modified Eagle's Medium (DMEM) containing 10%fetal calf serum, DMEM-Ham's F12 (3:1) medium containing 10% fetal calfserum, 5 μg/ml of transferrin, 5 μg/ml of insulin, 2 nMtri-iodotyrosine, 0.1 nM cholera toxin and 0.4 μg/ml of hydrocortisone,and keratinocyte growth medium (KGM) and DMEM containing 10% fetal calfserum mixed at a ratio of 1:1. Although varying according to theparticular type, the amount of matrix metalloproteinase inhibitor addedto these basal media is about 1 nmol/L to 10⁻² mole/L. In addition, theamount of matrix protein production promoting agent added to the basalmedium is about 1 ng/L to 1 g/L.

[0033] In the production of artificial skin of the present invention,contracted type I collagen gel containing human fibroblasts is firstallowed to stand undisturbed on a metal mesh. The contracted type Icollagen containing human fibroblasts can be prepared in, for example,the manner described below. After preparing a collagen solution in whichfibroblasts are suspended over ice, the collagen is gelled in a Petridish. Next, the gel is peeled from the walls of the Petri dish and thecollagen gel is contracted in a CO₂ incubator.

[0034] Next, epidermal cells, such as normal human epidermalkeratinocytes, are cultured on the above collagen gel to form epidermal.Formation of an epidermal layer by culturing epidermal cells can becarried out in the following manner. Contracted collagen gel is placedon a metal mesh, and a glass ring is placed on this gel. A suspension ofepidermal keratinocytes originating in human foreskin is placed in theglass ring while preventing liquid leaking. The keratinocytes areadhered in a CO₂ incubator, followed by removal of the ring. The abovemedium is filled to the boundary of the epidermal layer and cornifiedlayer is formed by continuing culturing while exposing the epidermallayer to the air.

[0035] According to this method, artificial-skin is obtained thatapproximates natural skin in which an adequate basement membrane isformed between a dermal layer and epidermal layer composed of contractedtype I collagen gel containing fibroblasts.

Skin Basement Membrane Formation Promoting Agent

[0036] According to the result of a study on basement membranestructural changes of facial skin for which cosmetics are primarily usedthat focused on a report of structural changes characterized byreduplication of the basement membrane accompanying aging as one of thechanges that accompany aging (Lavker, J., Invest. Dermal. 1979, 73,59-66), structural changes in the basement membrane begin to occur inthe late twenties and have been found to accumulate with age. Thesestructure changes of basement membrane are skin changes that precedeskin changes accompanying aging, such as the formation of wrinkles,hardening or decreased resiliency.

[0037] Thus, it is essential for expression of normal skin function thatepidermal basal cells be securely bound to the basement membrane, andpromotion of basement membrane formation is considered to be importantfor this. As a result of studying wrinkle formation inhibitors using anultraviolet radiation aged mouse model based on the presumption ofroutine ultraviolet rays, activity that inhibits wrinkle formation wasfound in drugs that are able to inhibit matrix metalloproteinase. Thus,agents that promote regeneration and repair of the basement membrane inthe skin are effective as inhibitors of skin aging.

[0038] Thus, according to the present invention, a skin activatingcomposition or basement membrane formation promoting artificial skinculturing liquid is newly provided that contains one type or a mixtureof two or more types of the above matrix metalloproteinase inhibitingcompound, or one type or a mixture of two or more types of this matrixmetalloproteinase inhibiting compound and one type or a mixture of twoor more types of a compound that demonstrates activity which increasesthe production of matrix protein, at a concentration sufficient forexhibiting effects that promote regeneration, repair and formation ofskin basement membrane. This skin activation refers to, for example, theprevention or improvement of decreased skin function accompanyingstructural changes in basement membrane caused by aging and so forth,and more specifically, skin wrinkling and hardening.

[0039] The above concentration sufficient for exhibiting effects thatpromote regeneration, repair and formation of skin basement membranerefers may vary according to the type of compounds used, othercomponents or vehicles used to prepare said composition, and theduration of use.

[0040] The skin basement membrane formation promoting agent of thepresent invention can contain active ingredient in the form of matrixmetalloproteinase inhibitor, or matrix metalloproteinase inhibitor andmatrix protein production promoting agent, at 0.000001 to 60 wt %, andpreferably 0.00001 to 60 wt %, relative to the composition.

[0041] The skin basement membrane stabilizing agent of the presentinvention can be in the form of an aqueous solution, oil, othersolution, milky liquid, cream, gel, suspension, microcapsules, powder,granules, capsules or solid, and after being prepared in any of theseforms according to methods which are themselves known, can be coated,adhered, sprayed, injected, consumed or inserted into the body in theform of a lotion, milky liquids, cream, ointment, paste, poultice,aerosol, injection, medication (including tablets, powders, granules,pills, syrups and troaches) or suppository. Among these preparationforms, skin topical preparations such as lotions, milky liquids, creams,ointments, pastes, poultices and aerosols are considered to bepreparation forms that are suitable for the composition of the presentinvention. The skin topical preparations described here includeprescription drugs, non-prescription drugs and cosmetics, and are to beused with the same meaning hereinafter.

[0042] Vehicles and fragrances routinely used when preparing suchcompositions, as well as oils, surfactants, antiseptics, sequesteringagents, water-soluble polymers, thickeners, powder ingredients,ultraviolet defensive agents, moisturizing agents, pharmacologicallyeffective ingredients, antioxidants, pH adjusters, cleaning agents,drying agents, emulsifiers and so forth can be suitably blended into thecomposition of the present invention. In the case of blending each ofthese ingredients into the skin activating composition of the presentinvention, it is necessary to blend them within a range that does notimpair the desired effect of the present invention.

[0043] Examples of the above oil include liquid oils, solid oils, waxes,hydrocarbon oils, higher fatty acids, higher alcohols, synthetic esteroils and silicones.

[0044] More specifically, examples of liquid oils include avocado oil,tsubaki oil, primrose oil, turtle oil, macadamia nut oil, corn oil, minkoil, olive oil, rape seed oil, egg yolk oil, sesame oil, persic oil,wheat germ oil, sasanqua oil, castor oil, linseed oil, safflower oil,cottonseed oil, perilla oil, soy bean oil, peanut oil, theine oil, kayaoil, rice bran oil, Chinese wood oil, Japanese wood oil, hohoba oil,germ oil, triglycerin, trioctanoic glycerin and triisopalmitic glycerin;examples of solid oils include cacao butter, coconut oil, horse tallow,hardened coconut oil, palm oil, beef tallow, goat tallow, hardened beeftallow, palm heart oil, pork -tallow, beef bone tallow, haze heart oil,hardened oil, beef leg tallow, haze wax and hardened castor oil;examples of waxes include beeswax, candelilla wax, cotton wax, carnaubawax, bayberry wax, tree wax, spermaceti, montan wax, bran wax, lanolin,kapok wax, lanolin acetate, liquid lanolin, sugar cane wax, isopropyllanolin fatty acid, hexyl laurate, reduced lanolin, jojoba wax, hardlanolin, shellac wax, POE lanolin alcohol ether, POE lanolin alcoholacetate, POE cholesterol ether, lanolin fatty acid polyethylene glycoland POE hydrogenated lanolin alcohol ether; and examples of hydrocarbonoils include liquid paraffin, ozokerite, squalene, pristan, paraffin,ceresin, squalene, Vaseline and microcrystalline wax.

[0045] Examples of higher fatty acids include lauric acid, myristicacid, palmitic acid, stearic acid, behenic (beheninic) acid, oleic acid,12-hydroxystearic acid, undecylenic acid, tolic acid, isostearic acid,linoleic acid, linolenic acid, eicosapentanoic acid (EPA) anddocosahexanoic acid (DHA).

[0046] Examples of higher alcohols include lauryl alcohol, cetylalcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleylalcohol, cetostearyl alcohol and other straight chain alcohols, as wellas monostearyl glycerin ether (vatyl alcohol), 2-decyltetradecinol,lanolin alcohol, cholesterol, phytosterol, hexyldodecanol, isostearylalcohol, octyldecanol and other branched chain alcohols.

[0047] Examples of synthetic ester oils include isopropyl myristate,cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butylstearate, hexyl laurate, myristyl myristate, decyl oleate, hexyldecyldimethyloctanoate, cetyl lactate, myristyl lactate, lanolin acetate,isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate,ethylene glycol di-2-ethylhexylate, dipentaerythritol fatty acid ester,n-alkylglyol monoisostearate, glycerin di-2-heptylundecanoate,trimethylolpropane tri-2-ethylhexylate, trimethylolpropanetriisostearate, pentaneerythritol tetra-2-ethylhexylate, glycerintri-2-ethylhexylate, trimethylolpropane triisostearate, cetyl-2-ethylhexanoate, 2-ethylhexyl palmitate, glycerin trimyristate,tri-2-heptylundecanoic glyceride, castor oil fatty acid methyl ester,oleic oil, cetostearyl alcohol, acetoglyceride, 2-heptylundecylpalmitate, diisobutyl adipate, N-lauroyl-L-glutamate-2-octyldodecylester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexylsebacate, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyladipate, diisopropyl sebacate, 2-ethylhexyl succinate, ethyl acetate,butyl acetate, amyl acetate and triethyl citrate.

[0048] Examples of silicones include dimethyl polysiloxane, methylphenylpolysiloxane, methylhydrogen polysiloxane and other linearpolysiloxanes, decamethyl polysiloxane, dodecamethyl polysiloxane,tetramethyltetrahydrogen polysiloxane and other cyclic polysiloxanes,and silicon resin and silicone rubber that form a three-dimensional meshstructure.

[0049] The skin basement membrane stabilizing agent of the presentinvention may contain anionic surfactant, cationic surfactant,amphoteric surfactant or nonionic surfactant and so forth either aloneor in combination.

[0050] Examples of anionic surfactants include fatty acid soaps such assoap base, sodium laurate and sodium palmitate; higher alkyl sulfateesters such as sodium lauryl sulfate and potassium lauryl sulfate; alkylether sulfate ester salts such as POE triethanol amine lauryl sulfateand POE sodium lauryl sulfate; N-acylsarcosinic acids such as sodiumlauroylsarcosine; higher fatty acid amide sulfonates such as sodiumN-myristoyl-N-taurine, sodium coconut oil fatty acid methyltauride andsodium laurylmethyltauride; phosphate esters such as POE sodium oleylether phosphate and POE stearyl ether phosphate; sulfosuccinates such assodium di-2-ethylhexylsulfosuccinate, sodium monolauroylmonoethanolamidepolyoxyethylene sulfosuccinate and sodium laurylpolypropylene glycolsulfosuccinate; alkylbenzene sulfonates such as linear sodiumdodecylbenzene sulfonate, linear dodecylbenzene sulfonatetriethanolamine and linear dodecylbenzene sulfonate; N-acylglutamatessuch as monosodium N-lauroylglutamate, disodium N-stearoylglutamate andmonosodium N-myristoyl-L-glutamate; higher fatty acid ester sulfateesters such as sodium hardened coconut oil fatty acid glycerin sulfate;sulfated oils such as Turkey red oil oil; as well as POE alkyl ethercarboxylic acid, POE alkylaryl ether carboxylic acid, α-olefinsulfonate, higher fatty acid ester sulfonate, secondary alcohol sulfateester, higher fatty acid alkylolamide sulfate ester, sodiumlauroylmonoethanolamide succinate, N-palmitoylaspartateditriethanolamine and sodium casein.

[0051] Examples of cationic surfactants include alkyl trimethyl ammoniumsalts such as stearyl trimethyl ammonium chloride and lauryl trimethylammonium chloride, dialkyl dimethyl ammonium salts such as distearyldimethyl ammonium chloride, alkyl pyridinium salts such as(N,N′-dimethyl-3,5-methylene piperidinium) chloride and cetyl pyridiniumchloride, alkyl quaternary ammonium salts, alkyl dimethylbenzyl ammoniumsalts, alkyl isoquinolinium salts, dialkyl morpholinium salts, POE alkylamines, alkyl amine salts, polyamine fatty acid derivatives, amylalcohol fatty acid derivatives, benzalkonium chloride and benzetoniumchloride.

[0052] Examples of amphoteric surfactants include imidazoline-basedamphoteric surfactants such as2-undecyl-N,N,N-(hydroxyethylcarboxymethyl)-2-imidazoline sodium and2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy-2-sodium salt, andbetaine-based amphoteric surfactants such as2-heptadecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine,lauryldimethylaminoacetate betaine, alkyl betaine, amidobetaine andsulfobetaine.

[0053] Examples of lipophilic nonionic surfactants include sorbitanfatty acid esters such as sorbitan monooleate, sorbitan monoisostearate,sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate,sorbitan sesqui dleate, sorbitan trioleate, penta-2-ethylhexylicdiglycerol sorbitan and tetra-2-ethylhexylic diglycerol sorbitan,glycerin polyglycerin fatty acids such as glycerin mono-cottonseed oilfatty acid, glycerin monoerucate, glycerin sesquiolate, glycerinmonostearate, glycerin α,α′-oleic pyroglutamate, glycerin monostearateand malic acid, propylene glycol fatty acid esters such as propyleneglycol monostearate, hardened castor oil derivatives, glycerin alkylether and polyoxyethylene-methylpolysiloxane copolymers.

[0054] Examples of hydrophilic nonionic surfactants include POE sorbitanfatty acid esters such as POE sorbitan monooleate, POE sorbitanmonostearate, POE sorbitan monooleate and POE sorbitan tetraoleate, POEsorbitol fatty acid esters such as POE sorbitol monolaurate, POEsorbitol monooleate, POE sorbitol pentaoleate and POE sorbitolmonostearate, POE glycerin fatty acid esters such as POE glycerinmonostearate, POE glycerin monoisostearate and POE glycerintriisostearate, POE fatty acid esters such as POE monooleate, POEdistearate, POE monodioleate and ethylene glycol distearate, POE alkylethers such as POE lauryl ether, POE oleyl ether, POL stearyl ether, POEbehenyl ether, POE 2-octyldodecyl ether and POE cholestanol ether, POEalkylphenyl ethers such as POE octylphenyl ether, POE nonylphenyl etherand POE dinonylphenyl ether, plurallonics such as pluronic, POE-POPalkyl ethers such as POE-POP cetyl ether, POE-POP 2-decyltetradecylether, POE-POP monobutyl ether, POE-POP hydrogenated lanolin and POE-POPglycerin ether, tetra POE-tetra POP ethylene diamine condensationproducts such as tetronic, POE castor oil/hardened castor oilderivatives such as POE castor oil, POE hardened castor oil, POEhardened castor oil monoisostearate, POE hardened castor oiltriisostearate, POE hardened castor oil monopyroglutamatemonoisostearate diester and POE hardened castor oil maleate, POEbeeswax-lanolin derivatives such as POE sorbitol beeswax, alkanol amidessuch as coconut oil fatty acid diethanol amide, lauric monoethanol amideand fatty acid isopropanol amide, POE propylene glycol fatty acidesters, POE alkyl amines, POE fatty acid amides, sucrose fatty acidesters, POE nonylphenyl formaldehyde compounds, alkylethoxydimethylamine oxides and trioleyl phosphates.

[0055] Examples of the above preservatives include methyl parabenzene,ethyl parabenzene and butyl parabenzene.

[0056] Examples of the above sequestering agents include sodium edetateand EDTA.

[0057] Examples of the above water-soluble polymers include naturalpolymers, semi-synthetic polymers, synthetic polymers and inorganicpolymers.

[0058] Examples of natural water-soluble polymers include plant polymerssuch as gum arabic, tragacanth gum, galactan, gua gum, carob gum, karayagum, carageenan, tamarind gum, xanthane gum, pectin, agar, queenseedmarmelo, algea colloid (brown algae extract), starch (rice, corn,potato, wheat) and glycyrrhizic acid, microbial polymers such asxanthane gum, dextran succinoglucan and pluran, and animal polymers suchas collagen, casein, albumin and gelatin.

[0059] Examples of semi-synthetic water-soluble polymers includestarch-based polymers such as dextrin, carboxymethyl starch andmethylhydroxypropyl starch, cellulose-based polymers such as methylcellulose, nitrocellulose, ethyl cellulose, methylhydroxypropylcellulose, hydroxyethyl cellulose, cellulose dimethyl dialkyl (12-20)ammonium sulfate, hydroxypropyl cellulose, sodium carboxymethylcellulose (CMC), crystalline cellulose and powdered cellulose,alginate-based polymers such as sodium alginate and propylene glycolalginate ester.

[0060] Examples of synthetic water-soluble polymers include vinyl-basedpolymers such as polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone, carboxyvinyl polymer and alkyl denatured carboxyvinylpolymer, polyoxyethylene-based polymers such as polyethylene glycol2000, 4000 and 6000, polyoxyethylene polyoxypropylene copolymer-basedpolymers, acrylic polymers such as sodium polyacrylate, polyethyleneacrylate and polyacrylamide, polyethylene imines and cationic polymers.

[0061] Examples of inorganic water-soluble polymers include bentonite,magnesium aluminum silicate, raponite, hectrite and silicic anhydride.

[0062] Examples of the above powder component include inorganic powderssuch as talc, kaolin, mica, sericite, muscovite, phlogopite, syntheticmica, lepidolite, biotite, lithia mica, vermiculite, magnesiumcarbonate, calcium carbonate, aluminum silicate, barium silicate,calcium silicate, magnesium silicate, strontium silicate, metaltungstate, magnesium, silica, zeolite, barium sulfate, baked calciumsulfate (baked gypsum), calcium phosphate, fluoroapatite,hydroxyapatite, ceramic powder, metal soap (zinc myristate, calciumpalmitate, aluminum stearate) and boron nitride, organic powders such aspolyamide resin powder (Nylon powder), polyethylene powder, polymethylmethacrylate powder, polystyrene powder, styrene and acrylic acidcopolymer resin powder, benzoguanamine resin powder,polytetrafluoroethylene powder and cellulose powder, inorganic whitepigments such as titanium dioxide and zinc oxide, inorganic red pigmentssuch as iron oxide (rouge) and iron titanate, inorganic brown pigmentssuch as γ-iron oxide, inorganic yellow pigments such as yellow ironoxide and ocher, inorganic black pigments such as black iron oxide,carbon black and low order titanium oxide, inorganic violet pigmentssuch as mango violet and cobalt violet, inorganic green pigments such aschromium oxide, chromium hydroxide and cobalt titanate, inorganic bluepigments such as ultramarine and Prussian blue, pearl pigments such astitanium oxide coated mica, titanium oxide coated bismuth oxychloride,titanium oxide coated talc, colored titanium oxide coated mica, bismuthoxychl bride and fish scale foil, metal powder pigments such as aluminumpowder and copper powder, zirconium, barium or aluminum lake organicpigments such as red no. 201, red no. 202, red no. 204, red no. 205, redno. 220, red no. 226, red no. 228, red no. 405, orange no. 203, orangeno. 204, yellow no. 205, yellow no. 401, yellow no. 404, red no. 3, redno. 104, red no. 106, red no. 227, red no. 230, red no. 401, red no.505, orange no. 205, yellow no. 4, yellow no. 5, yellow no. 202, yellowno. 203, green no. 3 and blue no. 1, natural pigments such aschlorophyll and β-carotene, and colorants such as titanium yellow,carsamine and saffron red.

[0063] Examples of the above ultraviolet defensive agents include both“ultraviolet absorbers”, which are substances that chemically absorbultraviolet rays, and “ultraviolet blockers”, which are substances thatscatter and reflect ultraviolet rays by physical action.

[0064] Namely, examples of long-wavelength ultraviolet (UVA) absorbersinclude anthranilic acid-based ultraviolet absorbers such as methylanthranilate and homomenthyl-N-acetylanthranilate, benzophenone-basedultraviolet absorbers such as 2,4-dihydroxybenzophenone,2,2-dihydroxy-4-methoxybenzophenone,2,2′-dihydroxy-4,4′-dimethoxybenzophenone, 2,2′,4,4′-tetrahydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone,2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, 4-phenylbenzophenone,2-ethylhexyl-4′-phenyl-benzophenone-2-carboxylate,2-hydroxy-4-n-octoxybenzophenone and 4-hydroxy-3-carboxybenzophenone,benzotriazole-based ultraviolet absorbers such as2,2′-hydroxy-5-methylphenylbenzotriazole,2-(2′-hydroxy-5′-t-octylphenyl)benzotriazole and2-(2′-hydroxy-5′-methylphenyl)benzotriazole, dianisoylmethane and4-methoxy-4′-t-butyldibenzoylmethane.

[0065] Among these long-wavelength ultraviolet absorbers,4-methoxy-4′-tert-butyldibenzoylmethane, 2-hydroxy-4-methoxybenzophenoneand 2-hydroxy-4-methoxybenzophenone derivatives such as2-hydroxy-4-methoxybenzophenone-5-sulfonate are preferable because theyare long-wavelength ultraviolet absorbers having superior safety andeffectiveness.

[0066] In addition, examples of medium-wavelength ultraviolet (UVB)absorbers include benzoic acid-based ultraviolet absorbers such asparaaminobenzoic acid (to be abbreviated as PABA), PABA monoglycerinester, N,N-dipropoxy PABA ethyl ester, N,N-diethoxy PABA ethyl ester,N,N-dimethyl PABA ethyl ester, N,N-dimethyl PABA butyl ester andN,N-dimethyl PABA amyl ester, salicylic acid-based ultraviolet absorberssuch as dipropylene glycol salicylate, ethylene glycol salicylate,myristyl salicylate, methyl salicylate, amyl salicylate, menthylsalicylate, homomenthyl salicylate, octyl salicylate, phenyl salicylate,benzyl salicylate and p-isopropanol phenyl salicylate, cinnamicacid-based ultraviolet absorbers such as octyl cinnamate,ethyl-4-isopropyl cinnamate, methyl-2,5-diisopropyl cinnamate,ethyl-2,4-diisopropyl cinnamate, methyl-2,4-diisopropyl cinnamate,propyl-p-methoxycinnamate, isopropyl-p-methoxycinnamate,isoamyl-p-methoxycinnamate, octyl-p-methoxycinnamate(2-ethylhexyl-p-methoxycinnamate), 2-ethoxymethyl-p-methoxycinnamate,cyclohexyl-p-methoxycinnamate, ethyl-α-cyano-β-phenylcinnamate,2-ethylhexyl-α-cyano-β-phenylcinnamate,glycerylmono-2-ethylhexanoyl-diparamethoxycinnamate, methoxycinnamateoctyl,3,4,5-trimethoxycinnamate-3-methyl-4-[methylbis(trimethylsiloxy)silyl]butyland p-dimethoxycinnamate monoethyl ester, camphor derivatives such as3-(4′-methylbenzylidene)-d,1-camphor, 3-benzylidene-d,1-camphor and5-(3,3-dimethyl-2-norvolnilidene)-3-penten-2-one, urocanic acid,urocanic ethyl ester, 2-phenyl-5-methylbenzooxazole and dibenzaladine.

[0067] Moreover, examples of ultraviolet blockers include titanium oxide(TiO₂), talc (MgSiO₂), carmine (FeO₂), bentonite, kaolin and zinc oxide(ZnO).

[0068] Examples of the above moisturizing agents include polyethyleneglycol, propylene glycol, glycerin, 1,3-butylene glycol, hexyleneglycol, xylitol, sorbitol, maltitol, chondroitin sulfuric acid,hyaluronic acid, mucoitin sulfuric acid, charoninic acid,atherocollagen, cholesteryl-12-hydroxystearate, sodium lactate, bileacid salt, d1-pyrrolidone carboxylate, short chain soluble collagen,diglycerin (EO) PO addition product, Chestnut rose Fruit extract,milfoil extract and melilote extract.

[0069] Examples of the above pharmacologically effective ingredientsinclude whiteners such as albutin, vitamin C and its derivatives, kojicacid placental extract, glutathione and saxifrage extract,antiphlogistics such as glycyrrhizic acid derivatives, glycyrrhetinicacid derivatives, salicylic acid derivatives, hinokitiol, zinc oxide andallantoin, activators such as royal jelly, actinolite, cholesterolderivatives and calf blood extract, circulation promoters such asnonylic urenylamide, nicotinic benzyl ester, nicotinic β-butoxyethylester, capsisin, zingerone, cantharidin, ichthammol, caffeine, tannicacid, α-borneol, nicotinic tocopherol, inositol hexanicotinate,cyclandelate, cinnarizine, tolazoline, acetylcholine, verapamil,cepharanthin and γ-olizanol, antiseborrheics such as sulfur andthianthol, and for various purposes, Phellodendron Bark extractcomponent, goldthread extract component, Lithospermum Root extractcomponent, peony extract component, gentian extract component, birchextract component, sage extract component, loquat extract component,carrot extract component, aloe extract component, mallow extractcomponent, iris extract component, grape extract component, Coix Seedextract component, sponge cucumber extract component, lily extractcomponent, saffron extract component, Cnidium Officinale Root extractcomponent, Ginger Root extract component, Saint John's-wort extractcomponent, Restharrow Root extract component, rosemary extractcomponent, garlic extract component, red pepper extract component, driedorange peel, Japanese angelica root and so forth, vitamin A substancessuch as retinol and retinol acetate, vitamin B₂ substances such asriboflavin butyrate and riboflavin adenine nucleotide, vitamin B2substances such as pyridoxine hydrochloride and pyridoxine dioctanoate,vitamin C substances such as L-ascorbic acid, L-ascorbate dipalmitateester, L-ascorbate-2-sodium sulfate, L-ascorbate phosphate ester anddipotassium DL-α-tocopherol-L-ascorbate phosphate diester, pantothenicacids such as calcium pantothenate, D-pantothenyl alcohol, pantothenylethyl ether and acetylpentothenyl ethyl ether, vitamin D substances suchas ergocalciferol and cholecalciferol, nicotinic acids such as nicotinicacid, nicotinic amide and benzyl nicotinate, vitamin E substances suchas α-tocopherol and DL-α-tocopherol succinate, and other vitamins suchas vitamin P and biotin.

[0070] Furthermore, these pharmacological components can be widelyblended within a range over which the desired pharmacological effect ofthe present invention is not impaired by that blending.

[0071] The composition of the present invention prepared in this mannerprevents decreased function accompanying structural changes of basementmembrane, and promotes activation of the skin.

EXAMPLES

[0072] The following provides a more detailed explanation of the presentinvention through its examples.

Example 1 Production of Artificial Skin Using Matrix MetalloproteinaseInhibitor

[0073] Collagen gel was obtained by preparing ,10 ml of a collagensolution of suspended fibroblasts (0.3 to 1×10⁵ cells/ml) originating inhuman dermis (product I-AC of Koken Co., Ltd. was used for the collagen)on ice followed by gelling the collagen at 37° C. in a 60 mm Petri dish.Next, the gel was peeled from the walls of the Petri dish and thecollagen gel was contracted in a CO₂ incubator.

[0074] This contracted collagen gel equivalent to dermis was placed on ametal mesh and a glass ring (inner diameter: 12 mm) was placed over thisgel. 0.4 ml of a suspension of epidermal keratinocytes (1×10⁶/ml) (mixedmedium of KGM-DMEM containing 5% fetal calf serum (1:1)) were placedinside the glass ring to prevent leakage of liquid. After adheringkeratinocytes in a CO₂ incubator overnight, the ring was removed on thefollowing day. The above medium was filled to the boundary of theepidermal layer and culturing was continued while exposing the epidermallayer to air to produce a skin model having a stratified epidermisexhibiting corneal layer formation.

[0075] After inoculating the epidermic cells, the medium was changed tothat containing either (1) 10 μM CGS23027A (matrix metalloproteinaseinhibitor) or (2) 300 μM MMP-inhibitor(p-NH₂-Bz-Gly-Pro-D-Leu-D-Ala-NHOH (FN-437) (BBRC, 1994, 199, p.1442-1446) (acquired from Cabbiochem. Novabiochem Corporation) (matrixmetalloproteinase inhibitor)) starting in the first week, and thenreplaced with medium containing the same type and same concentration ofmatrix metalloproteinase inhibitor every 2-3 days after that andcultured for 2 weeks. In addition, (3) similar culturing was carried outwithout adding matrix metalloproteinase inhibitor for use as a control.

[0076] Those results are shown in FIG. 1. As is clear from the figure,although a basement membrane-like structure was not observed directlybeneath the basal cells of the epidermis in control (3), in the case ofadding matrix metalloproteinase inhibitors (1) CGS27023A compound or (2)MMm-inhibitor, promotion of basement membrane formation was clearlyobserved.

[0077] A similar experiment was carried out for extracts of the plantsindicated below instead of the above matrix metalloproteinase inhibitorcompounds to confirm promotion of basement membrane formation.

[0078] Similar effects were confirmed for Thymus serpyllum L., Valerianafauriei Briquet or other similar plants (Valerianaceae), Diospyros kakiThunberg (Ebenaceae), Astragalus sinicus Linne (Leguminosae), Crataeguscuneata Siebold et Zuccarini (Rosaceae), Paeonia suffruticosa Andrews(Poeonia montan Sims) (Paconiaceae), Thea sinensis Linne var. assamicaPierre (Thcaccae), Eucalyptus globulus Labillardiere or its similarplants (Myrtaceae), Potentilla tormentilla Schrk (Rosaceae), Tiliacorda:a Mill., Tilia platyphyllus Scop., Tilia europaea Linne(Tiliaceae), Betula alba Linne (Betulaceze), Origanum majorana L.,Uncaria gambir Roxburgh (Rubiaceae), Juglans regia Linne var. sinensisDe Candolie or its similar plants (Juglandaceae), Sophora flavescensAiton (Leguminosae), Sanguisorba officinalis Linne (Rosaceae), Hypericumperforatum Linne or Hypericum erectum Thunberg (Guttiferae), Theasinensis Linne (Theaceae), Curcuma longa L (Zingiberaceae), purifiedextracts of Curcuma longa L including Symplocos racemosa and Cyperusrotundus, Cyperpus scariosus, Gaultheria fragrantissima, Acaciafornensia, Terminalia chebula, Ficus bengalensis, Cassia fistula Linn,Lyonia ovalifolia, Calophyllum inophyllum and Ficus religiosa.

Example 2 Production of Artificial Skin Using Both MatrixMetalloproteinase Inhibitor and Matrix Protein Production PromotingAaent

[0079] Although the same method as Example 1 was repeated, the followingsubstances were used as test substances.

[0080] (1) 10 μM matrix metalloproteinase inhibitor CGS27023A only;

[0081] (2) 10 μM matrix metalloproteinase inhibitor CGS27023A and 10ng/ml of matrix metalloproteinase protein production promoting agent,Transforming Growth Factor α (TGFa);

[0082] (3) 10 μM matrix metalloproteinase inhibitor CGS27023A and 10ng/ml of matrix protein production promoting agent, Transforming GrowthFactor β1 (TGFb1);

[0083] (4) 10 μM matrix metalloproteinase inhibitor CGS27023A, 10 ng/mlof matrix protein production promoting agent, Transforming Growth Factorα (TGFa) and 10 ng/ml of Transforming Growth Factor β1 (TGFb1); and

[0084] (5) culturing of a control to which none of the above were added.

[0085] The formed artificial skin was stained by hematoxylin-eosin (H &E) and immunostaining (using anti-laminin 5 (LN5) antibody, anti-type IVcollagen (IVC) antibody, and anti-type VII collagen (VIIC) antibody).Those results are shown in FIG. 2, FIG. 3 and Table 1. Furthermore, theresults for H & E and LN5 are shown in FIG. 2, while the results for IVCand VIIC are shown in FIG. 3.

[0086] In the control, adhesion of epidermis/dermis joined portions wasweak, the epidermis easily peeled from the dermis and there were voidsobserved in the tissue. In contrast, in the group treated with matrixmetalloproteinase inhibitor CGS27023A, adhesion between the epidermisand dermis was clear. In addition, in the case of adding matrix proteinproduction promoting agent, Transforming Growth Factor α (TGFa),Transforming Growth Factor β1 (TGFb1) or both (TGFa+TGFb1) in additionto matrix metalloproteinase inhibitor CGS27023A, staining of type VIIcollagen in particular was promoted significantly, thereby confirmingthat adhesion between the epidermis and dermis was further promoted.TABLE 1 Effects of Various Drugs on Basement Membrane Formation in aSkin Model Deposition of basement membrane component Basement TreatmentType IV Type VII membrane conditions Laminin 5 collagen collagenformation Non-treatment + + − − control group CGS27023A ++ ++ + +CGS27023A + ++ ++ ++ ++ soybean lecithin CGS27023A + ++ ++ ++ TGFαCGS27023A + ++ ++ ++ TGFβ1 CGS27023A + ++ ++ ++ TGFα + TGFβ1

[0087] Culturing was carried out in the same manner as above using thefollowing plant extracts instead of the above matrix metalloproteinaseinhibitor CGS27023A.

[0088]Thymus serpyllum L., Valeriana fauriei Briquet or other similarplants (Valerianaceae), Diospyros kaki Thunberg (Ebenaceae) , Astragalussinicus Linne (Leguminosae), Crataegus cuneata Siebold et Zuccarini(Rosaceae), Paeonia suffruticosa Andrews (Poeonia montan Sims)(Paconiaceae), Thea sinensis Linne var. assamica Pierre (Thcaccae),Eucalyptus globulus Labillardiere or its similar plants (Myrtaceae),Potentilla tormentilla Schrk (Rosaceae), Tilia corda:a Mill., Tiliaplatyphyllus Scop., Tilia europaea Linne (Tiliaceae), Betula alba Linne(Betulaceze), Origanum majorana L., Uncaria gambir Roxburgh (Rubiaceae),Juglans regia Linne var. sinensis De Candolie or its similar plants(Juglandaceae), Sophora flavescens Aiton (Leguminosae), Sanguisorbaofficinalis Linne (Rosaceae), Hypericum perforatum Linne or Eypericumerectum Thunberg (Guttiferae), Thea sinensis Linne (Theaceae), Curcumalonga L (Zingiberaceae), purified extracts of Curcuma longa L includingSymplocos racemosa and Cyperus rotundus, Cyperus scariosus, Gaultheriafragrantissima, Acacia fornensia, Terminalia chebula, Ficus bengalensis,Cassia fistula Linn, Lyonia ovalifolia, Calophyllum inophyllum and Ficusreligiosa.

[0089] As a result, results were obtained that were similar to the caseof using CGS27023A as matrix metalloproteinase inhibitor. In addition,similar results were also obtained for Symplocos racemosa, a purifiedextract of Curcuma longa L.

[0090] Moreover, a structure resembling a basement membrane wascontinuously observed directly beneath the basal cells in the case ofusing 30 μg/ml of soy bean lecithin instead of Transforming GrowthFactor for the matrix protein production promoting agent. Those resultsare shown in FIG. 4.

Example 3 Preparation of Artificial Skin Culture Medium

[0091] Matrix metalloproteinase inhibitor CGS27023A or MMP-inhibitorwere added to a concentration of 10 μM to basal medium comprising a 1:1mixture of KGM (keratinocyte growth medium), typically used to cultureepidermic cells, and Dulbecco's Modified Eagle's Medium containing 10%fetal calf serum, typically used to culture fibroblasts. Furthermore,KGM was prepared by adding HEPES (6.7 g/L), NaHCO₃ (1.2 g/L), insulin (5mg/L), transferrin (10 mg/L), hydrocortisone (0.5 mg/L) andphosphorylethanolamine (14.1 mg/L) to MCDB153 medium (10.93 g/L) andadjusting the pH to 7.4 followed by the addition of 20 g of bovinepituitary extract (Far East Pharmaceutical).

Example 4 Preparation of Artificial Skin Culture Medium

[0092] At least one type of Transforming Growth Factor a (TGFa) orTransforming Growth Factor β1 (TGFb1) were added as matrix proteinproduction promoting agent to the artificial skin culture medium inExample 3 to a concentration of 10 ng/ml.

[0093] In addition, soy bean lecithin was added at 30 μg/ml in place ofthe above TGFa or TGFb1.

Preparation Example 1 Cream

[0094] Polyoxyethylene (addition of 20 moles) cetyl 1.0 alcohol etherMethylphenyl polysiloxane (20 cs) 2.0 Liquid paraffin 3.02-hydroxy-4-methoxybenzophenone 5.0 Active ingredient 0.2 Propyleneglycol 5.0 Glycerin 2.0 Ethyl alcohol 15.0 Carboxyvinyl polymer 0.3Hydroxypropyl cellulose 0.1 2-aminomethylpropanol 0.1 Antiseptic Assuitable Fragrance As suitable Ion exchanged water As suitable

Production Method

[0095] Propylene glycol, glycerin, ethyl alcohol, carboxyvinyl polymer,hydroxypropyl cellulose and 2-aminomethylpropanol were added to ionexchanged water followed by heating to 70° C. (aqueous phase).

[0096] Methylphenyl polysiloxane, liquid paraffin, polyoxyethylene cetylalcohol ether, antiseptic, 2-hydroxy-4-methoxybenzophenone, activeingredient (such as lipidure and/or CGS27023A) and fragrance were mixedand prepared to 70° C. (oily phase).

[0097] After gradually adding the aqueous phase to the oily phase,pre-emulsifying and obtaining homogeneous emulsified particles using ahomomixer, the emulsion was deaerated and cooled to obtain a cream.

Preparation Example 2 Cream

[0098] Polyoxyethylene (addition of 20 moles) cetyl 1.0 alcohol etherMethylphenyl polysiloxane (20 cs) 2.0 Liquid paraffin 3.02-hydroxy-4-methoxybenzophenone 5.0 Active ingredient 0.2 Propyleneglycol 5.0 Glycerin 2.0 Ethyl alcohol 15.0 Carboxyvinyl polymer 0.3Hydroxypropyl cellulose 0.1 2-aminomethylpropanol 0.1 Antiseptic Assuitable Fragrance As suitable Ion exchanged water As suitable

Production Method

[0099] Propylene glycol, glycerin, ethyl alcohol, carboxyvinyl polymer,hydroxypropyl cellulose and 2-aminomethylpropanol were added to ionexchanged water followed by heating to 70° C. (aqueous phase).

[0100] Methylphenyl polysiloxane, liquid paraffin, polyoxyethylene cetylalcohol ether, antiseptic, 2-hydroxy-4-methoxybenzophenone, activeingredient (such as lipidure and/or CGS27023A) and fragrance were mixedand prepared to 70° C. (oily phase).

Preparation Example 3 Milky Liquid

[0101] Wt % Cetyl alcohol 1.0 Beeswax 0.5 Vaseline 2.0 Squalene 6.0Dimethyl polysiloxane 2.0 Ethyl alcohol 5.0 Glycerin 4.0 1,3-butyleneglycol 4.0 Active ingredient 0.1 Tranexamic acid 1.0 Polyoxyethylene(10) monooleic ester 1.0 Glycerol monostearic ester 1.0 Queenseedextract (5% aqueous solution) 20.0 Antiseptic As suitable Fragrance Assuitable Ion exchanged water As suitable

Production Method

[0102] Glycerin and 1,3-butylene glycol were added to ion exchangedwater, mixed and heated to 70° C. (aqueous phase). Cetyl alcohol,beeswax, Vaseline, squalene, dimethyl polysiloxane, active ingredient(such as lipidure or CGS27023A), tranexamic acid, polyoxyethylene (10)monooleic ester, glycerol monostearic ester and antiseptic were preparedin the form of a mixture and heated to 70° C. (oily phase). After addingthe aqueous phase to the oily phase and performing preliminaryemulsification, adding and stirring queenseed extract and ethyl alcoholand obtaining homogeneous emulsified particles using a homomixer, theemulsion was deaerated, filtered and cooled to obtain a milky liquid.

Preparation Example 4 Milky Liquid

[0103] Wt % Cetyl alcohol 1.0 Beeswax 0.5 Vaseline 2.0 Squalene 6.0Dimethyl polysiloxane 2.0 Ethyl alcohol 5.0 Glycerin 4.0 1,3-butyleneglycol 4.0 Active ingredient 0.1 Tranexamic acid 1.0 Polyoxyethylene(10) monooleic ester 1.0 Glycerol monostearic ester 1.0 Queenseedextract (5% aqueous solution) 20.0 Antiseptic As suitable Fragrance Assuitable Ion exchanged water As suitable

Production Method

[0104] Glycerin and 1,3-butylene glycol were added to ion exchangedwater, mixed and heated to 70° C. (aqueous phase). Cetyl alcohol,beeswax, Vaseline, squalene, dimethyl polysiloxane, active ingredient(such as lipidure or CGS27023A), tranexamic acid, polyoxyethylene (10)monooleic ester, glycerol monostearic ester and antiseptic were preparedin the form of a mixture and heated to 70° C. (oily phase). After addingthe aqueous phase to the oily phase and performing preliminaryemulsification, adding and stirring queenseed extract and ethyl alcoholand obtaining homogeneous emulsified particles using a homomixer, theemulsion was deaerated, filtered and cooled to obtain a milky liquid.

[0105] After gradually adding the aqueous phase to the oily phase,pre-emulsifying and obtaining homogeneous emulsified particles using ahomomixer, the emulsion was deaerated and cooled to obtain a cream.

1. A skin basement membrane formation promoting agent comprising amatrix metalloproteinase inhibitor.
 2. A skin basement membraneformation promoting agent comprising matrix metalloproteinase inhibitorand matrix protein production promoting agent.
 3. An artificial skinformation promoting agent comprising matrix metalloproteinase inhibitor.4. An artificial skin formation promoting agent comprising matrixmetalloproteinase inhibitor and matrix protein production promotingagent.
 5. A production method of artificial skin comprising addingmatrix metalloproteinase inhibitor to artificial skin formation medium.6. A production method of artificial skin comprising adding matrixmetalloproteinase inhibitor and matrix protein production promotingagent to artificial skin formation medium.